Preparation of spherical shaped mycelial pellets

ABSTRACT

Spherical shaped mycelial pellets having a support core and which are suitable for biocatalytic conversion of organic compounds are produced by dissolving a cellulose derivative in a solvent to form a solution, mixing spores of a mycelial microorganism with the solution, precipitating the cellulose derivative to form porous beads containing the spores, and incubating the beads in a culture medium. Alternatively, the pellets may be produced by dissolving agar in water, mixing the agar solution with spores of a mycelial microorganism, precipitating the agar to form beads containing the spores and incubating the beads in a culture medium.

BACKGROUND OF THE INVENTION

The invention relates generally to the art of biological catalyticreactor systems with mycelia of microorganisms, especially mycelialfungi, and more particularly to mycelial pellets having a support coreand use thereof for biocatalytic conversions, as well as the preparationof the biologically active mycelial pellets.

Microorganisms have long been employed in the biocatalytic conversion oforganic compounds. Exemplary of such conversions, to mention but a few,include conversion of simple sugars to useful products such as alcoholsand organic acids; production of enzymes; synthesis of antibiotics andisomerization of sugars. Thus, the conversion of various sugars has tosome degree involved the use of fungi, but such use has been generallylimited to an unsupported vegetative mass. U.S. Pat. No. 4,127,447describes a biocatalytic reaction involving a packed column packed witha support material to which have been attached the needed anaerobicmicroorganisms. U.S. Pat. No. 4,090,022, describes the use of porouscellulose beads to which active biological agents (e.g. enzymes) may beattached by means of chemical bonding. In each case the active agent isbonded to the support, thus necessitating preparation of the support forattachment of the desired agent.

The use of mycelial fungi in biocatalytic systems has been limited dueto the difficulty in handling such masses. While supported mycelia wouldbe desirable, no satisfactory method has been developed prior to thepresent invention, and methods to date require preparation of thesupport surface in order to attach the active agent. It is known thatsome mycelial fungi can form mycelial pellets in a conventional shakingculture. While such a pellet form is more desirable than the unshapedvegetative mass, the pellets suffer a number of disadvantages. Forexample, if placed in a column reactor, under flow pressure, the pelletmay collapse and plugging will occur. Furthermore, handling and recoveryof these pellets are hampered due to the weak physical properties of thepellets.

Since the discovery of the present invention, K. Gbewonyo and D. I. C.Wang have reported the growth of mycelial microorganisms on sphericaldiatomaceous beads (Abstract of Papers presented at 178th ACS Meeting,Washington, D.C., Sept. 10-13, 1979--American Chemical Society Divisionof Microbial and Biochemical Technology). The report describes thegrowth of Penicillium chrysogenum on porous celite beads resulting inthe projection of hyphae outwards from the bead surface. No surfacelayer of structural integrity is reported.

In accordance with the present invention, we have discovered usefulspherical shaped mycelial pellets which possess an inner rigidstructural core which is surrounded by a porous webbed layer havingstructural integrity of a mycelial microorganism. The porous webbedlayer thus forms a spherical encasement of structural integrity aboutthe rigid core.

Accordingly, it is the primary object of the present invention toprovide mycelial pellets having a core support surrounded by a porouswebbed layer of structural integrity of a mycelial microorganism.

It is a further object of the present invention to provide an improvedmeans for carrying out the biocatalytic conversions of organiccompounds.

These and other objects of the present invention will become moreapparent from the discussion which follows.

SUMMARY OF THE INVENTION

When mycelial microorganisms, such as fungi, grow in a liquid medium,their mycelia form a loose cotton-like mass. As used herein, mycelialmicroorganisms are defined as those living microorganisms, thevegetative portion of which forms filamentous hyphae. Such mycelialmicroorganisms include various fungi (including some yeasts), bacteria(e.g., Actinomyces) and algae such as blue-green algae. Of particularinterest are mycelial fungi, especially from the genus Rhizopus andMucor.

Some mycelial fungi can form pellets when they are grown in a circularaction shaking incubator. The formation of pellet increases the celldensity and facilitates the separation of the cell mass from the liquidproducts. Unfortunately not every fungus is able to form such mycelialpellets in a circular action shaking incubator. It is desirable toprepare the mycelia in a pellet form for use in a fermentation process,especially in a continuous fermentation process. In a continuousfermentation process, the flow properties and the cell density determinethe efficiency of the fermentation process. The fungal mycelial pelletsprepared by conventional circular action shaking cultures are loose andfragile when they are packed in a column reactor. They are easilydeformed under small pressure which may result from a flow of thereaction substrate.

The present invention also provides a method for the preparation ofmycelial microorganism pellets from all types of mycelialmicroorganisms, particularly fungi, with a rigid physical support core.Those fungi which are incapable of forming mycelial pellets inconventional shaking cultures can form mycelial pellets by the presentmethod.

The pellets thus prepared may if desired possess a rigid physicalsupport core, and thus exhibit good flow properties when they are usedin a continuous fermentation reactor. The diffusion of the nutrients andthe products into and out of the conventional mycelial pellets is ratherslow. The supported mycelial pellets prepared by the present inventiongrow into round pellets with a porous webbed layer of mycelia generallyhaving a thickness of from about 0.1 mm to about 5 mm (preferably 1 to 3mm), the webbed layer thereby forming a spherical encasement ofstructural integrity about the rigid core. The distance between thesupport core and the mycelial layer will vary from 0 to 1 cm (preferablyabout 5 mm) depending upon the initial spore concentration andincubation time. The pellets with such structural mycelial layers alloweasy diffusion of nutrients into and passage of products out of thepellets.

The mycelial pellets prepared according to the present invention possessan outer mycelial layer having a highly texturized structure which isresistant to shear force. Mycelial pellets prepared by conventionalmethod have loose mycelial structure that would be sheared off at highflow rates experienced in a continuous reactor. The mycelial pelletsmade according to this invention can be regenerated even at a high flowrate in the reactor because the "seed" spores still exist inside thebead core.

One embodiment of the present invention relating to the preparation ofmycelial pellets is in part based on the procedure of making cellulosebeads as described in U.S. Pat. No. 4,090,022 (the entire contents ofwhich is incorporated herein by reference). The procedure is as follows:

(a) Dissolving a hydrolyzable cellulose derivative in an inert organic,water-miscible solvent to form a solution having a density greater thanthat of a precipitation solution;

(b) Mix mycelial microorganisms spores (e.g. fungal spores) with thecellulose derivative solution;

(c) Distribute the solution in the form of droplets into a precipitationsolution or cool air to form porous beads containing said spores;

(d) Separating the precipitated beads from the precipitation solution ifused;

(e) Eliminate undesirable bacteria by chemical sterilization or washingwith sterilized water thoroughly;

(f) If cellulose beads are desired, the beads can be regenerated beforeincubation of the beads;

(g) Incubate the beads containing the spores in a liquid culture mediumwith agitation (e.g. in a circular action shaking incubator).

Thus, the process for the preparation of biologically active mycelialpellets having a physical support generally comprising the steps of:

(a) dissolving a hydrolyzable cellulose derivative in an inert organic,water-miscible solvent to form a solution having a density greater thanthat of a precipitation solution;

(b) mixing mycelial microorganism spores with said solution anddistributing the resulting solution in the form of droplets into aprecipitation solution or cool air whereby said cellulose derivative isprecipitated in the form of uniformly porous beads, said beadscontaining said spores;

(c) separating the precipitated beads from said precipitation solutionif used;

(d) washing the separated porous beads to eliminate undesirable bacteriaand facilitate incubation of said spores;

(e) incubating said separated porous beads in a liquid culture mediumfor a period of time sufficient to produce a round pellet characterizedby a rigid porous bead core surrounded by a porous webbed layer havingstructural integrity of a mycelial microorganism, depending upon theinitial spore concentration and length of incubation, the space betweenthe core and layer may be substantially void, with a number of myceliaforming a filamentous connection between the core and webbed layer.

The inert organic water miscible solvent may be a single liquid orcombination of liquids as described in the aforementioned U.S. Pat. No.4,090,022 as well as its predecessor, U.S. Pat. No. 4,063,017, theentire contents of which are also incorporated herein by reference.

As used herein, the term "precipitation solution" is defined as a liquidsolution which is a non-solvent for the cellulose derivative and ismiscible with the above inert organic, water-miscible solvent. By meansof illustration, the precipitation solution may be water or an aqueoussolution. The precipitation solution is therefore miscible with solventcomponents. Thus, it will be appreciated that when one dissolves thecellulose derivative in the organic solvent and subsequently adds a dropof the resulting solvent solution to the precipitation solution, thecellulose derivative will coagulate and precipitate out due to the phaseinversion which the cellulose derivative undergoes thereby forming thedesired porous cellulose bead.

When employing an aqueous precipitation solution, one may suitable useas solvent component (a) a member from the group consisting of acetone,formamide a mixture of acetone and methanol, methyl acetate, a mixtureof methylene dichloride and methanol, methyl ethyl ketone and dimethylsulfoxide. The solvent component (b) may thus be suitably chosen from amember selected from the group consisting of dimethyl sulfoxide,formamide, methyl acetate, cyclohexanone, methylene dichloride, ethylenedichloride, a mixture of methylene dichloride and methanol and a mixtureof ethylene dichloride and methanol.

The preferred precipitation solution into which the solution ofcellulose derivative is to be distributed generally consists of water,but may be an aqueous solution which contains suitable amounts ofnon-ionic or ionic surfactants to reduce the surface tension thereof andfacilitate formation of the porous beads.

Alternatively, one may prepare the supported pellets by the steps:

(a) Providing a porous core support (e.g. prepare cellulose or cellulosederivative beads);

(b) Absorb the fungal spores with these beads; and

(c) Incubating the beads with agitation (e.g. in a circular actionshaking incubator).

The physical and mechanical properties of the mycelial pellets can beimproved by cross-linking the mycelium or impregnating the mycelialpellet with cellulose or cellulose derivatives or other inexpensivepolymers. Suitable cross-linking agents which could be used includeamong others glutaraldehyde and diisocyanate.

One may also prepare biologically active mycelial pellets with agar, inaccordance with a further embodiment of the present invention. Agar is acarbohydrate and its solution melts at 90° C., solidifying at about 45°C. Because it is inert to most microorganisms, agar is used to solidifyliquid culture media. These characteristics of agar can be utilized as asupporting core material to trap spores of mycelial microorganisms andproduce mycelial pellets.

In general agar may be used as a supporting material in preparingmycelial pellets by:

(a) dissolving agar in hot water to form an agar solution;

(b) cooling the agar solution to a temperature of greater than 45° C.,but below the temperature at which the spores added in step (c) would bekilled (generally from between about 45° and 80° C. preferably about 50°C.);

(c) mixing the agar solution of step (b) with spores of a mycelialmicroorganism and dispersing the resulting mixture in the form ofdroplets into cool air or an aqueous precipitation solution at atemperature below 45° C. whereby said agar is precipitated in the formof uniform beads containing said spores;

(d) separating the agar beads from the precipitation solution if used,or alternatively, one may employ the same solution for bothprecipitation and incubation; and

(e) incubating the agar beads with agitation (e.g. in a circular actionshaking incubator) for a period of time sufficient to produce a roundpellet characterized by a rigid round agar core surrounded by porouswebbed layer of mycelial having structural integrity of a microorganism.

Incubation of the mycelial spores according to the present invention iscarried out with agitation using nutrients, pH and temperatureparameters which are conventional for the microorganisms employed.Generally, the nutrient medium is an aqueous solution containing asource of the essential elements (i.e. carbon and nitrogen) to supportthe vegetative growth of the microorganism hyphae. Generally glucose andammonia may be employed in the nutrient medium. The pH of the medium mayvary generally from about 3 to 8, but preferably ranges from about 4.5to 6.5. The temperature for incubation may also vary widely depending onthe microorganism (e.g. 0° to 70° C., preferably about 20° to 38° C.).

The amount of spores to be added to the core forming medium is notcritical, but should be at a level sufficient to provide at least onespore for each bead core produced or impregnated. Generally, aconcentration of at least about 10⁶ spore per 100 ml of core formingmedium is sufficient to provide an optimum level of germination.

The mycelial pellets of the present invention may be furthercharacterized as having a substantially void space between the coresurface and the webbed mycelial layer depending upon initial sporeconcentration and incubation time. The space may be as great as 1 cmwhen measured from the core surface to the webbed layer depending uponthe number of spores present in the bead core and time of incubation. Byreferring to a substantially void space, it is to be understood thatsome of the hyphae resulting from the vegetative growth of the sporesmay remain anchored to the spore thereby forming a network offilamentous connections between the bead core and webbed mycelial layerencompassing the core.

The following materials and procedures were employed in evaluating thepresent invention.

ISOLATION AND MAINTENANCE OF THE MUCORACEOUS FUNGI

Several Chinese yeast preparations were obtained from various locationson the island of Taiwan. The dry circular cake of Chinese yeast cultureswas first cracked and broken into a fine powder. This powder wassuspended in sterile water and plated onto potato dextrose agar (PDA)plates containing 0.02% rose-bengal (Sigma). Rose-bengal inhibits thegrowth of yeasts and bacteria, but it inhibits the growth offast-growing molds to a lesser extent. The PDA rose-bengal plates wereincubated at 30° C. for 48 hours before the mycelial edge of a fungalcolony was transferred. Single sporangiospores were then isolated.Several isolates were identified as Rhizopus species, and the rest wereidentified as Mucor species.

Cultures were maintained at 4° C. on PDA slants and transferred monthly.

Inoculum and Cultural Conditions

Both growing and non-growing mycelial systems were used. The growingmycelial system was as follows

Small amounts of sporangiospore suspension were inoculated into 250 ml.Erlenmeyer flasks containing 100 ml of basic salts medium (BSM)consisting of 2.0 gm. KH₂ PO₄, 1.4 gm (NH₄)₂ SO₄, 0.3 gm. urea, 0.3 gmCaCl₂, 0.3 gm. MgSO₄.7H₂ O, 1.0 gm. peptone (Difco), 10.0 gm. eitherglucose or xylose, 1.0 mg. Fe⁺⁺, 0.5 mg. Mn⁺⁺, 0.8 mg. Zn⁺⁺, and 0.5 mg.Co⁺⁺, each per liter of culture medium, and finally 0.011 M in sodiumcitrate buffer, pH 5.8. These cultures were incubated at 30° C.overnight on a reciprocal shaker and then harvested by filtration.

The non-growing mycelial system was as follows

Fresh mycelia, harvested from the growing mycelial system, wereintroduced into a 250 ml. Erlenmeyer flask containing 100 ml. of BSMwith either 20.0 gm. glucose, or 10 gm. xylose per 100 ml., andincubated at 30° C. in a New Brunswick psycrotherm incubator shaker.These cultures were kept under nitrogen gas for the appropriate periodof time. Unless otherwise indicated, sugars and BSM broth wereautoclaved separately and mixed before inoculation.

Determination of Dry Weight of Mycelia

Cultures were passed through lint-free Miracloth filters (ChicopeeMills, Inc.), the retained mycelia washed with distilled water, squeezeddry and allowed to dry in an 80° C. oven overnight.

The following examples are offered in order to more fully describe theinvention, but are not to be construed as limiting the scope thereof.

EXAMPLE 1

Five grams of cellulose acetate were dissolved in 40 ml of solvent (6parts acetone and 4 parts dimethyl sulfoxide) to form a 10% (W/v)solution. Spores of Rizopus spp. 0.1 gm were mixed into the celluloseacetate evenly. With a hand-made spray gun the cellulose solution wasthen sprayed into a water tank. The cellulose acetate beads coagulatedin the water tank and precipitated to the bottom of the tank. The beadswere collected and washed with tap water and then washed with sterilizedwater thoroughly. The washed beads were then incubated in a selectiveliquid medium in a circular action shaking incubator. After three daysthe mycelial pellets were formed.

EXAMPLE 2

After the beads were formed and collected as in Example 1, the sporecontaining cellulose acetate beads were regenerated to cellulose beadsin a NaOH solution (1 N) for one hour and then washed thoroughly withsterilized water. After incubation for three days in the circular actionshaking incubator, the mycelial pellets were formed.

EXAMPLE 3

The procedures of Examples 1 and 2 were repeated except that the fungalspores were from Aspergillus niger. Mycelial pellets having a rigid coreencompassed by a porous layer of A. niger of structural integrity wwereobtained as in the foregoing examples.

EXAMPLE 4

Beads of cellulose acetate and cellulose were prepared, and thereafterthe fungal spores were added to the beads. After stirring and washingsome spores were absorbed in the beads. Incubation of both types ofbeads resulted in the formation of mycelial pellets having the structureof the present invention, although the porous webbed mycelial layer waslooser than the resulting mycelial layer in Examples 1 to 3.

EXAMPLE 5

1 gm of agar was dissolved in 50 ml of water in a boiling water bath.The agar solution was cooled to 50° C. 50 mg of spores from Mucor sp wasadded to the solution and mixed. The solution was sprayed into a coldwater tank (15° C.). Upon contacting the surface of the water, the agarsolidified as droplets and sank to the bottom. After the sporecontaining agar beads were collected and washed thoroughly withsterilized water, the beads were incubated in a liquid medium in acircular-action shaking incubator for 3 days and the mycelial pelletswere formed.

EXAMPLE 6

The beads containing spores from Rizopus spp. were prepared as inExample 5. Mycelial pellets were formed.

EXAMPLE 7

The spores of Rizopus spp. were mixed with agar beads prepared as inExample 5 except that spores were not added to the agar solution. Afterwashing, a small quantity of spores were absorbed on the surface of theagar beads. The beads were incubated in a liquid medium in a circularaction shaking incubator, and the mycelial pellets formed in three days.However, the structure of the mycelial wall of the pellets was looserthan that in Examples 5 and 6.

The mycelial pellets of the present invention lend themselves to a widevariety of applications for the biocatalytic conversion of organiccompounds. Thus, depending upon the conversion desired and selection ofmycelial microorganism, one may carry out such conversions as:

(a) production of alcohols and/or organic acids from sugars;

(b) production of specific enzymes;

(c) synthesis of antibiotics (e.g. pencillin); and

(d) isomerization of organic compounds (e.g. sugars).

The mycelial pellets of the present invention are particularly wellsuited for use in column reactors as they provide enhanced flow and masstransfer properties over columns of packed mycelia or unsupportedmycelial pellets. The mycelial pellets could also be used to provide acolumn reactor containing the pellets and which is suitable for use inhome brewing operations.

Such a column containing mycelia capable of fermenting sugar solutionsto alcohol could be stored at low temperatures (or alternatively themycelial pellets could be freeze dried) which when used is merelyconnected to a sugar solution (e.g. grape juice or cider) forpreparation of an alcoholic drink.

The invention, in its broadest aspects, is not limited to the specificdetails shown and described, but departures may be made from suchdetails within the scope of the accompanying claims without departingfrom the principles of the invention. Furthermore, the invention maycomprise, consist, or consist essentially, of the hereinbefore recitedmaterials and steps.

What is claimed is:
 1. A process for the preparation of biologicallyactive mycelial pellets having a physical support core which comprisesthe steps of:(a) dissolving a hydrolyzable cellulose derivative on aninert organic, water-miscible solvent to form a solution having adensity greater than that of a precipitation solution; (b) mixing sporesof a mycelial microorganism with said solution and distributing theresulting solution in the form of droplets into a precipitation solutionor cool air whereby said cellulose derivative is precipitated in theform of uniformly porous beads, said beads containing said spores; (c)separating the precipitated beads from said precipitation solution ifused; (d) incubating the precipitated porous beads in an aqueous culturemedium for a period of time sufficient to produce a round pelletcharacterized by a rigid spherical porous bead core surrounded by aporous integral webbed layer, said layer forming a spherical encasementof structural integrity about the rigid core and being composed offilamentous hyphae of a mycelial microorganism.
 2. A process accordingto claim 1, wherein said cellulose derivative is selected from the groupconsisting of cellulose acetate, cellulose triacetate, and cellulosenitrate.
 3. A process according to claim 1 wherein said microorganism isa fungi selected from the genus Rhizopus or Mucor.
 4. A processaccording to claim 1 wherein the precipitated porous beads are washedwith sterile water prior to incubating.
 5. A process according to claim4 wherein the incubation is carried out with agitation.
 6. A processaccording to claim 1 wherein the distributing is accomplished byspraying.
 7. A process according to claim 1 wherein said precipitationsolution is selected from the group consisting of water, hexane,cyclohexane, octane, benzene and mixtures of water and ethanol ormethanol.
 8. A process according to claim 7 wherein said precipitationsolution is water.
 9. A process according to claim 1 wherein saidsolvent is a mixture of:(a) a member from the group consisting ofacetone, a mixture of acetone and methanol or ethanol, methyl acetate, amixture of methylene dichloride and methanol, methyl ethyl ketone,formamide and dimethyl sulfoxide; and (b) a member from the groupconsisting of dimethyl sulfoxide, formamide, methyl acetate,cyclohexanone, methylene dicholoride, ethylene dichloride, a mixture ofmethylene dicholoride and methanol, and a mixture of ethylenedicholoride and methanol.
 10. A process according to claim 9 whereinsaid solvent is dimethyl sulfoxide, formamide or methyl acetate.
 11. Aprocess for the preparation of biologically active mycelialmicroorganism pellets which comprise the steps of:(a) dissolving agar inhot water to form an agar solution; (b) cooling the agar solution to atemperature of greater than 45° C. but below the temperature at whichthe spores added in step (c) would be killed; (c) mixing the agarsolution of step (b) with spores of a mycelial microorganism anddispersing the resulting mixture in the form of droplets into cool airor an aqueous precipitation solution at a temperature below 45° C.whereby said agar is precipitated in the form of uniform beadscontaining said spores; (d) incubating the agar beads in an aqueousculture medium for a period of time sufficient to produce a round pelletcharacterized by a rigid round agar core surrounded by a porous integralwebbed layer, said layer forming a spherical encasement of structuralintegrity about the rigid core and being composed of filamentous hyphaeof a mycelial microorganism.
 12. A process according to claim 11 whereinthe mycelial spores are fungi of the genus Rhizopus or Mucor.
 13. Aprocess according to claim 11 wherein the agar beads are washed withsterile water prior to incubating.
 14. A process according to claim 11or claim 12 wherein dispersing the said mixture is accomplished byspraying.
 15. A process according to claim 11 wherein the cooling instep (b) is carried out at a temperature ranging between about 45° to80° C.
 16. A process according to claim 15 wherein the medium solutionis cooled to about 50° C.
 17. A process according to claim 11 whereinthe aqueous precipitation solution is the aqueous culture medium.
 18. Aprocess according to claim 11 wherein the droplets are dispersed byspraying into cool air.
 19. A process according to claim 11 wherein thedroplets are dispersed by spraying into cool water.
 20. A sphericalshaped mycelial pellet produced according to the process of claim
 1. 21.A pellet according to claim 20 wherein said cellulose derivative isselected from the group consisting of cellulose acetate, cellulosetriacetate and cellulose nitrate.
 22. A spherical shaped mycelial pelletproduced according to the process of claim
 11. 23. A pellet according toclaim 1 or claim 22 wherein said microorganism is a fungi of the genusRhizopus, Mucor, Aspergillus, Pencillium or Trichoderma.
 24. A pelletaccording to claim 20 or claim 22 having a core diameter ranging fromabout 0.1 mm to 1.0 cm and a layer thickness of about 1 mm to 5 mm. 25.A method for the biocatalytic conversion of organic compounds whichcomprises reacting said compounds in the presence of the mycelial pelletof claim 20 or claim
 22. 26. A method according to claim 25 wherein acarbohydrate material is converted to alcohol and said microorganism isa fungi of the genus Rhizopus, Mucor, or mixtures thereof.
 27. A methodaccording to claim 25 wherein said conversion is carried outcontinuously in a column reactor containing said mycelial pellets.
 28. Abiocatalytic column reactor containing the mycelial pellet of claim 20or claim 22.